Project Summary As a hallmark of alcohol use disorder (AUD), the withdrawal syndrome includes both negative symptoms like anxiety and motivational symptoms such as craving for alcohol, which promotes relapse and alcohol seeking. Understanding how alcohol withdrawal changes brain physiology is an important step towards developing effective treatments to reduce alcohol abuse. The ventral tegmental area (VTA) is an important brain area that projects to components of the extended amygdala, including the nucleus accumbens, prefrontal cortex, amygdala and hippocampus. Changes in the physiology of VTA neurons induced by withdrawal may underlie the alcohol-seeking during AUD. The sensitivity of neurons of the VTA to inhibition by gamma aminobutyric acid (GABA) is decreased during alcohol withdrawal but normalized by histone deacetylase (HDAC) inhibitors, indicating epigenetic changes induced by withdrawal in the VTA may be amenable to pharmacological manipulation. Whole genome sequencing and molecular studies of this project identified a cluster of genes that encode cholesterol synthesis pathway enzymes that showed decreased expression during alcohol withdrawal, and are functionally relevant to decreased GABA sensitivity in VTA neurons during withdrawal. The proposed project plans to further characterize the role of specific epigenetic modifications in withdrawal-induced regulation of genes responsible for cholesterol synthesis, and the effect of these changes in withdrawal-induced GABA hyposensitivity and drinking behaviors. By using electrophysiological, behavioral, and state of the art molecular biological methods such as chemogenetics and CRISPR technology, we anticipate achieving the following goals: 1) to reveal novel epigenetic marks and changes in gene expression associated with chronic alcohol exposure and withdrawal with whole genome approaches, 2) to examine whether decreased histone acetylation and increased histone methylation reduce expression of cholesterol synthesis enzymes in the VTA during withdrawal in a cell-type specific manner, and probe the role of cholesterol synthesis enzyme genes in withdrawal-related drinking behavior and GABA hyposensitivity, 3) to determine whether targeted epigenetic intervention counteracts withdrawal-induced phenotypes by using CRISPR to prevent decreases in histone acetylation on promoters of key cholesterol synthesis enzyme genes, and 4) to translate to post-mortem human alcoholic VTA the epigenetic dynamics and expression of genes related to GABA hyposensitivity that have been identified in the rat VTA. Ultimately, these studies are needed to understand epigenetic adaptation of VTA neurons involved in the positive affective state during alcohol withdrawal, and, with the other components of this Center, will provide a great deal of information on epigenetic mechanisms involved in withdrawal-induced brain changes, and possible pharmacological approaches toward more effective treatment of AUD.